Conditioning of bioactive glass surfaces in protein containing solutions

ABSTRACT

Conditioning of the surface of silica-based glass or ceramic by differential immersion in a serum protein-containing solution, and the resultant microporous Ca—P surface layer having serum-protein like organic molecules, as defined herein intermingled throughout, is described.

REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of application Ser. No.08/647,171, filed May 9, 1996, (currently pending) which is incorporatedherein by reference in its entirety.

GOVERNMENT RIGHTS

The invention disclosed herein was made with Government support. TheUnited States government may have rights to certain aspects of thisinvention.

FIELD OF THE INVENTION

This invention relates to materials comprising silica-based glass orceramic compositions having been treated to form a microporouscalcium-phosphate (Ca—P) surface layer having serum-protein like organicmolecules, as defined herein, intermingled throughout and, optionally,other biologically active molecules intermingled throughout and/oradsorbed thereon.

BACKGROUND OF THE INVENTION

In U.S. Pat. No. 5,204,106, issued to Schepers et al., (herebyincorporated by reference) the implantation of bioactive glass granuleshaving the composition: 45% SiO₂, 24.5% Na₂O, 24.5% CaO and 6% P₂O₅, anda size range of 280-425 μm in diameter into the jaw of beagle dogs isdisclosed. With particles of this size range, internal pouches formed ineach of the particles and, subsequently, osteoprogenitor cellsdifferentiated into osteoblasts within the pouches, actively laying downbone tissue. Next, bone tissue proliferated from the excavations,surrounded the particles and connected with bone tissue being formedaround neighboring particles. Bone tissue was formed within theparticle, without being bridged to the lingual or buccal bone plates. At3 months, bone had grown throughout the surgical defects treated withglass granules. A similar phenomenon was not observed with particles ofa larger size range, i.e., ˜480-800 micrometers, or a smaller sizerange, i.e., ˜210-300 micrometers.

Glass granules of narrow size range (300-360 μm) were used in a clinicaltrial in humans. Schepers et al., “Bioactive Glass Particles of NarrowSize Range: A New Material for the Repair of Bone Defects,” ImplantDentistry, 2(3):151-156, 1993, incorporated herein by reference. In thisclinical study, 87 patients and 106 maxillo facial defects were treated.At 3 months, the application sites had fully solidified. At six months,no radiological difference between the defect sites and the surroundingbone could be discerned.

It was subsequently discovered that particles in the size range 200-300micrometers effected the same results if implanted into sites exhibitinga reduced metabolic state, particularly as compared to themaxillo-facial sites. Such sites are found, for example, in theappendicular skeleton, and in certain disease states. U.S. applicationSer. No. 08/268,510, hereby incorporated by reference.

The in vivo event which initiates the reactions leading to the formationof bone throughout the defects is an excavation of the particles. Theexcavation is the result of physico-chemical reactions taking place inthe glass, as well as a cell-mediated resorption of the internalreaction layer. Bioactive glass reacts at its surface with the formationof two reaction layers: a silica gel below the surface and a Ca—P richlayer at the surface.

It has now been discovered that differential immersion of othergeometric forms of melt- or sol-gel-derived silica-based glass orceramic in aqueous solutions containing serum-protein like organicmolecules, as defined herein, results in a microporous surface layer ofCa—P having the serum-protein like organic molecules intermingledthroughout. It is expected that silica-based glass or ceramic soconditioned will have an improved performance in vitro and in vivo,e.g., in relation to cell phenotype expression and attachment of cellsin general. Prior treatments did not achieve this surface layer.

SUMMARY OF THE INVENTION

According to the present invention, silica-based glasses or ceramics areconditioned to achieve a microporous Ca—P surface layer havingserum-protein like organic molecules, as defined herein, intermingledthroughout and, optionally, biologically active molecules intermingledthroughout and/or adsorbed thereon.

In one aspect, the present invention relates to a method for modifyingthe surface of silica-based glasses or ceramics by differentialimmersion in aqueous solutions containing serum-protein like organicmolecules to achieve a microporous Ca—P surface layer havingserum-protein like organic molecules intermingled throughout.

In yet another aspect, the present invention relates to a materialcomprising silica-based glasses or ceramics having a microporous Ca—Psurface having serum-protein like organic molecules herein intermingledthroughout, prepared by differential immersion of the silica-basedglasses or ceramics in aqueous solutions containing serum-protein likeorganic molecules.

In a further aspect, the present invention relates to a materialcomprising silica-based glasses or ceramics having a microporous Ca—Psurface layer having serum-protein like organic molecules intermingledthroughout.

BRIEF DESCRIPTION OF THE DRAWING

FIGS. 1a-c depict the changes in Si, Ca, and P content, respectively,during immersion of sol-gel derived particles as a function of glasscomposition.

FIG. 2 is a Scanning Electron Micrograph depicting a hollowcalcium-phosphate particle formed from sol-gel derived glass.

FIG. 3 depicts changes in Si content during immersion of sol-gel derivedparticles of S70 glass composition as a function of particle size.

FIG. 4 depicts the effect of serum on amount of Si released for S70sol-gel derived glass particles.

FIGS. 5a-c are SEM, EDXA, and FTIR, respectively, depicting the changesin morphology, composition and structure, respectively, of the surfacesfollowing differential immersion in serum-containing solution.

FIGS. 6a-c are SEM, EDXA, and extended carbon peaks of a cross-sectionof a granule following differential immersion in serum-containingsolution.

FIGS. 7a-c are SEM, EDXA, and FTIR, respectively, depicting the changesin morphology, composition and structure, respectively, of the surfacesfollowing differential immersion in serum-free solution.

FIGS. 8a-c are SEM, EDXA, and FTIR, respectively, depicting the changesin morphology, composition, and structure, respectively, of the surfacesfollowing integral immersion in serum-free solution followed byimmersion in serum-containing solution.

FIG. 9 is an RBS of a sample following integral immersion in serum-freesolution followed by immersion in serum-containing solution.

FIGS. 10a-d are SEM, EDXA (b and c), and FTIR, respectively, depictingthe changes in morphology, composition, and structure, respectively, ofthe surfaces following integral immersion in serum-containing solution.

DETAILED DESCRIPTION

The materials treated according to the present invention can be used ina variety of applications including, without limitation, to fill bonydefects in sites present throughout the body of vertebrates forstimulating growth and repair. The materials can be used for the invitro growth of bone and other tissues. The materials can also be usedin vitro or in vivo with anchorage dependent cells or at sites with suchcells. The materials are formed as a result of conditioning silica-basedglass materials containing calcium and phosphate in vitro.

As used herein, “silica-based” refers to the inclusion of a siliconoxide in the composition of the glass. Other oxides may also be present.

“Bioactive” as used herein refers to a bone bioactive material having acalcium phosphate rich layer present, or which develops duringappropriate in vitro or in vivo conditions. As observed by Pereira etal., J. of Biomed. Mat. Res., (1994) 28:693-698 (incorporated herein byreference), pure silica gel having a porous hydrated layer is able toinduce a carbonated hydroxyapatite layer when soaked in a simulated bodyfluid containing calcium and phosphate ions. Pure silica hydrogelsproduced using TEOS and drying temperatures of around 400° C. wereimmersed in simulated body fluids having different magnesium, calcium,and phosphate ions. It was reported that apatite nucleation inductionperiods were decreased with the addition of small amounts of calcium andphosphate ions to the fluids, as well as increase in pH. Li et al., J.Appl. Biomater., (1993) 4:221-229 and Li et al., J. Amer. Ceram. Soc.,(1993) 75:2094-2097 (both incorporated herein by reference).

Melt-derived silica-based, calcium and phosphate containing materials tobe conditioned according to the present invention can be preparedfollowing, for example, the procedure disclosed in U.S. Pat. No.5,204,106, hereby incorporated by reference. The composition for themelt-derived glass can be as follows (in weight percent): SiO₂-40-60;Na₂O-10-32 CaO-10-32 and P₂O₅-0-12. The composition for the melt-derivedglass can also be SiO₂-39-70; Na₂-0-25; CaO-8-20; and P₂O₅-0-6. Brink,Maria, J. Biomed. Mat. Res., 36: 109-117, 1997, incorporated herein byreference. Other compounds, and oxides, may also be present, such asdescribed in P. Ducheyne, “Bioglass coatings and bioglass composites asimplant materials,” J. Biomed. Mat. Res., 19:273-291, 1985, incorporatedherein by reference.

The sol-gel derived materials can be prepared following, for example,the procedures disclosed in U.S. Pat. No. 5,591,453, hereby incorporatedby reference. The compositional range for the sol-gel derived glass canbe as follows (in weight percent): SiO₂-60-100; CaO-0-40; and P₂O₅-0-10.

The in vitro excavation of the silica-based, Ca—P containing glassgranules and transformation into Ca—P shells disclosed in priorapplication Ser. No. 08/647,171 was achieved by differential immersionof the glass granules in, inter alia, serum-containing solutions whichprovoked the dissolution of silicon from the glass matrix. Similarconditioning can be applied to other geometric forms of melt- andsol-gel-derived bioactive glasses, including, but not limited to, disks,blocks, monoliths, fibers, coatings, and rods, resulting in a novelsurface. Differential immersion can be achieved by periodic solutionexchange in a system in which the solution is static, or by use of adynamic system in which the immersion solution is continuously suppliedas a flow of fresh solution. See, U.S. Pat. No. 5,002,890, issued toDennis R. Morrison on Mar. 26, 1991 for an example of how to achievedynamic fluid replenishment in, that case, tissue culture.

The immersion conditions such as, for example, solution composition,duration, number of solution exchanges, material weight to solutionvolume ratio (W/V), can vary to a great degree as long as the formationof the calcium phosphate layer is perpetuated. The appropriateconditions to achieve the invention described herein can be readilydetermined by one skilled in the art.

The immersion conditions for the in vitro excavation were previouslyselected to allow the following events to occur: 1) formation of asurface Ca—P layer; and 2) continuous dissolution of the silica-network,remaining under the Ca—P layer, until about 80 to 100% of the originalSi content in the glass is dissolved. In order to stimulate thecontinuous silica network dissolution, the solution used for theimmersion must remain undersaturated with respect to soluble silica.Events 1) and 2) can be achieved by consecutively different solutions.For example, in the case of a glass composed of 100% silica, acalcium-phosphate surface layer must first be formed. This can beaccomplished by immersing the glass in solutions saturated in silicon,such as described in U.S. application Ser. No. 08/647,007, herebyincorporated by reference.

The immersion conditions for conditioning the silica-based glass andceramics according to the present invention are similar. The exchange ofsolution perpetuates the formation of the microporous Ca—P surface layerhaving serum-protein like organic molecules, as defined herein,intimately intermingled throughout.

Once this microporous Ca—P surface layer is formed, subsequentimmersions can be performed in aqueous solutions containing otherbiologically active molecules, with or without the serum-protein likeorganic molecules, as defined herein, present. The subsequent immersionscan be performed in an integral mode, as defined herein, and areperformed for a time sufficient to allow adsorption of the biologicallyactive molecules onto the surface of the microporous Ca—P surface layer,including within the pores. For example, immersion times of from about 5minutes to about 160 hours are contemplated.

The following reactions are involved in the transformation of themelt-derived glass: hydrolysis and formation of a silica-gel layer;migration of Ca and PO₄ ions from the bulk through the Si-gel to thesurface; accumulation of the ions at the surface and formation of a Ca—Prich surface; continuous growth of the Si-gel and Ca—P rich layers; andcontinuous silica dissolution.

The following reactions are involved in the transformation of sol-gelderived glass granules: loss of soluble silica along with migration ofCa and PO₄ ions through highly porous glass to the surface; leaching ofthe ions to a solution along with partial accumulation at the surface;formation of a CaP-surface layer; growth of the CaP-layer along withcontinuous silica dissolution.

Some aqueous solutions which can be used include, but are not limitedto, the following:

a) water;

b) ion-free, Tris buffer, with an initial pH of from about 6.8 to about8.0—for melt-derived bioactive glass;

c) phosphate buffer solution with an initial pH of from about 6.8 toabout 8.0; and

d) Tris or phosphate buffer solutions containing varying concentrationsof Ca and PO₄ (HPO₄ or H₂PO₄) with or without addition of varyingconcentrations of other ions including, but not limited, to Na, K, Cl,CO₃, and Mg, or their combination.

Solution d) is used when the glass or ceramic is 100% silicon oxide. Theimmersion solution must also contain serum-protein like organicmolecules, as defined herein, which result in the formation of amicroporous Ca—P surface layer. The immersion solution can also containbiologically active molecules as define herein, and/or such moleculescan be adsorbed after formation of the microporous Ca—P surface layer.

With increasing stability of the silica-based glass—such as increasingthe amount of network former SiO₂, increasing the amount of networkmodifiers that render the network more stable (see Ducheyne, supra), or,in the case of melt-derived glass, replacing Na₂O with a more stablealkali oxide—the duration of treatment and/or the number of solutionexchanges will have to be increased, or the material size will have tobe reduced. The presence of serum-protein like organic molecules in theimmersion solution decreases the dissolution rate. A possibleexplanation is that the adsorption of the proteins on the surface of theglass modifies the surface of the glass in contact with solution.

The dissolution of various forms of solid silica in aqueous solutionshas been the focus of several studies. O'Connor et al., “The kineticsfor the solution of silica in aqueous solutions,” J. Phys. Chem.,62:1195-8, 1958; and Iler, R. K., The Chemistry of Silica: Solubility,Polymerization, Colloid and Surface Properties, and Biochemistry, Wiley,N.Y., 1979. It has been established that any form of solid silica incontact with aqueous solutions dissolves into monosilicic acid Si(OH)₄until the solution reaches saturation. By exchanging the immersionsolution such that saturation is never reached, the diffusion process isfavored and thus dissolution is enhanced. The in vitro dissolutionbehavior during the differential immersion is expected to be similar tothat observed in vivo, during which the body fluid is continuouslyreplenished.

“Microporous Ca—P surface layer” as used herein refers to a surfacelayer containing calcium and phosphorous. Other compounds, for example,silicon, may also be present.

As used herein, the term “about” means approximately ±10%, except whenreferring to immersion times, in which case about means ±a day or two.

“Serum-protein like organic molecules”, as defined herein, refers toorganic molecules having the same effect as serum proteins followingdifferential immersion of silica-based glass or ceramic therein, i.e.,resulting in the formation of a microporous Ca—P surface layer.Serum-protein like organic molecules according to the present inventioncan be readily determined by those skilled in the art following thedisclosure herein. Serum-protein like organic molecules produced bysynthetic means, including genetic engineering, are included in thepresent invention, as are derivatives containing the active domains ofsaid organic molecules, e.g., the binding domains of attachmentmolecules. When present during differential immersion, serum-proteinlike organic molecules slow down the Ca—P precipitation which protectsthe underlying glass network from degradation during dissolution. TheCa—P layer is also microporous and, thus, there is not sufficientprotection of the underlying glass network, and dissolution of siliconand formation of a Ca—P layer at the surface continues with solutionreplenishment. Combinations of serum-protein like organic molecules arealso included.

The term “bony defect”, as used herein, refers to regions necessitatinggrowth or repair including, but not limited to, fractures, areas oferosion or degradation, osteolysis, holes resulting from removal ofscrews and pins, replacements, periodontal applications, anddeterioration of bone due to old age or disease.

As used herein, “biologically active molecules” are defined as thoseorganic molecules having an effect in a biological system, whether suchsystem is in vitro, in vivo, or in situ. Biologically active moleculesinclude, but are not limited to, the following categories: growthfactors, cytokines, antibiotics, anti-inflammatory agents, analgesicsand other drugs, and cell attachment molecules. It is contemplated thatmany serum-protein like organic molecules will also function asbiologically active molecules, and vice versa.

The term “antibiotic” includes bactericidal, fungicidal, andinfection-preventing drugs which are substantially water-soluble suchas, for example, gentamicin, vancomycin, penicillin, and cephalosporins.

The term “growth factors” refers, without limitation, to factorsaffecting the function of cells such as osteogenic cells, fibroblasts,neural cells, endothelial cells, epithelial cells, keratinocytes,chondrocytes, myocytes, cells from joint ligaments, and cells from thenucleus pulposis. Platelet derived growth factors (PDGF), thetransforming growth factors (TGF-β), insulin-like growth factors (IGFs),fibroblast growth factors (FGFs), and the bone morphogenetic proteins(BMPs) are examples of growth factors encompassed in the variousgeometric forms according to the present invention.

The term “cell attachment molecules” as used herein includes, but is notlimited to, fibronectin, vitronectin, collagen type I, osteopontin, bonesialoprotein, thrombospondin, and fibrinogen. Such molecules areimportant in the attachment of anchorage-dependent cells to the tissuematrix or to implant materials.

The term “intermingled throughout” as used herein means that themolecules, e.g., serum-protein like organic molecules, as definedherein, are distributed throughout the microporous Ca—P surface layerand are not just adsorbed on or embedded in on its surface.

The term “microporous” as used herein refers to a pore size of fromabout 0.1 to about 10 μm.

The term “macroporous” as used herein refers to a pore size of fromabout 40 to about 1000 μm.

The term “dense” as used herein in reference to a glass or ceramic meanssubstantially non-porous.

Previously, we proposed to treat the bioactive glass with a two steptreatment (see U.S. Pat. No. 5,643,789). We treated bioactive glassbecause we found that creating a Ca—P surface on which proteins wereadsorbed stimulated the expression of the osteoblast phenotype. However,the Ca—P layer which is created is relatively thin, in contrast to thelayer one observes on bioactive glass after in vivo implantation. Inaddition, the Ca—P and protein are not mixed together as would be thecase when the layer is formed in vivo. In this previously inventedtwo-step conditioning treatment, the first step of the treatment isperformed in protein-free medium and quickly leads to the formation of aprotective Ca—P film which prevents the corrosion reactions fromproceeding deeper and deeper into the material. Further, since theproteins are only adsorbed in a second step, the protein layer isadsorbed only on top of the Ca—P layer.

Immersing the bioactive glass in a solution containing serum withoutchanging the solution during the immersion period—i.e., an integralimmersion as the term is used herein—did not produce the desiredcomposite organic-inorganic layer—i.e., a layer with intermingled Ca—Pand serum-protein like organic molecules. Further, we found that thekinetics of the formation of the Ca—P surface reaction layers wereextremely slow in comparison with its formation in an electrolytesolution without serum. Others before us had also used tissue culturesolutions to study reaction layer formation on bioactive glasses.

In the present invention, we disclose a microporous Ca—P surface layerintermingled with serum-protein like organic molecules. Furthermore,this invention includes the methodology to obtain relatively thickCa—P-serum-protein like organic molecule layers, which can be digestedby cells. The thicker Ca—P-serum-protein like organic molecule layer fordigestion by cells is expected to have a beneficial effect.

We propose that the carbonated hydroxyapatite layer typically observedon bioactive glass after implantation is formed as a result of activityof bone cells adhered onto the initial reaction layer. This reasoningfollows from two sets of experiments in our laboratory. We showed that,prior to the formation of a carbonated hydroxyapatite layer, anamorphous Ca P layer, intermingled with Si, forms. Performing Fouriertransform infrared spectroscopy on two sets of glass samples afteridentical experimental conditions except for the presence of neonatalrat calvaria osteoblasts, we also found either no evidence of carbonatedhydroxyapatite (no cells present), or very clear evidence (withosteoblasts) (El-Ghannam et al., J.Biomed.Mat.Res., 29: 359-370,1995).Furthermore, using energy dispersive X-ray analysis, we measuredsignificant differences in elemental concentration on one sample aftertwo days of osteoblast culture between areas covered by extracellularmatrix elaborated by the cells, and those areas not yet covered(El-Ghannam et al., Biomat., 18:295-303 (1997). Prominent Ca, P and Sipeaks were evident under the extracellular matrix. Areas not covered bymatrix revealed small Si and P peaks and a pronounced Ca peak.Noteworthy, it is not just the interaction of the initial surfacereaction layer with cells, but also the intriguing presence of Si inthose areas where there is marked cellular activity.

Furthermore, having the serum-protein like organic moleculesintermingled throughout the surface is expected to be beneficial aswell. It has been previously reported that adsorbed, nonspecificproteins at a bone site are regularly eliminated from the ceramicsurface due to cellular degradation (Rohanizadeh et al., Bioceramics,10:27-30, 1997). Accordingly, a thick layer of Ca—P having proteinsintermingled throughout will stimulate additional cellular activity.

We have now found that if we continuously replenish the immersionsolution in which the surface reaction layer is formed, we perpetuatethe reaction at the glass or ceramic surface and continue to form anincreasingly thicker layer of microporous Ca—P intermingled with organiccompounds of the immersion solution. Furthermore, the microporous Ca—Psurface layer forms even when the glass or ceramic to be treated is notitself porous, i.e., is dense.

Replenishing the solution can be achieved by immersing the glass objectsin a volume of solution in, for example, a vial or receptacle andexchanging the solution from time to time either through a partial ortotal solution exchange. Alternatively, the glass or ceramic to besurface-treated can also be placed in a system that permits continuousflow of the treatment solution past the glass surfaces, such as arediscussed above.

The surface layers and the methods for making these are extremely usefulin any application where cells make contact with the material by virtueof the stimulatory nature of the surfaces on cell function. By nowhaving a surface that is a continuous mixture of Ca—P and serum-proteinlike organic molecules, there is a continual intra-and extra-cellulareffect arising from both the chemical components of the mineral phase ofbone as well as the serum-protein like organic molecules. This effectwill greatly stimulate tissue engineering procedures in vitro and invivo. Bone tissue engineering procedures will particularly benefit fromthese new and unexpected findings. Further, any anchorage dependent cellcan be placed in contact with the newly developed surface reactionlayer.

In our invention there is the added advantage that silicon isincorporated throughout the surface reaction layer in tandem with theserum-protein like organic molecules. No other treatment leads to thesimulanteous incorporation of both. Data using our prior two-steptreatment indicates the presence of prominent Si and P peaks, inaddition to a Ca peak, only under extracellular matrix elaborated byosteoblast cells in culture on bioactive glass. El-Ghannam et al., J.Biomed. Mat. Res., 29:359-370 (1995) and El-Ghannam et al., Biomat.,18:295-303 (1997).

Others have suggested that silicon is associated with calcium in anearly stage of bone formation. Carlisle found that silicon is requiredfor normal growth and development in the chick. E. M. Carlisle, Science,167:279-280 (1970) and E. M. Carlisle, Science, 178:619-621 (1972).Schwarz et al. reported that silicon deficiency in the rat resulted indepressed growth and skull formation. Schwarz et al., Nature,239:333-334 (1972). Later, Carlisle reported that silicon's primaryeffect is on the matrix, i.e., that silicon is required for collagen andglycosaminoglycan formation. Carlisle also noted that additional supportfor silicon's metabolic role in connective tissue was provided by thefinding that silicon is a major ion of osteogenic cells and is presentin especially high concentrations in the metabolically active state ofthe cell and that, further, silicon reaches relatively high levels inthe mitochondria of these cells. E. M. Carlisle, Ciba FoundationSymposium, 121:123-139 (1986).

As is disclosed below, the composition and morphology of the surface ofthe invention are different from that formed by immersion in aserum-free medium, followed by an immersion in serum. Using the methodaccording to the invention, we found that the reaction surface comprisesa microporous Ca—P surface layer having serum-protein like organicmolecules, intermingled throughout. The surface layer formed in serumhas a lower Ca/P ratio (about 1.2-1.3) than the surface formed inserum-free solutions (>1.45) and contains significant amounts of Si ofwhich it was previously suggested to have an effect on bone cellfunction. The invention lies in the achievement of this reactionsurface.

The method to create these beneficial reaction layers on silica-basedglass or ceramic, e.g., bioactive glass, is typically as follows. Thesolution containing the serum-protein like organic molecules cancomprise serum. The serum source can be, without limitation thereto,human, bovine, porcine, etc. Differential immersion (immersion withsolution exchange at designated time periods) in serum-containing,buffered (pH 7-7.6) solutions such as Tris or phosphate bufferedsolution, either ion-free or containing ions similar to those found inhuman plasma. Serum content can vary from about 1% to about 100%,preferably from about 8% to about 100%. The total protein content inserum generally can vary from about 3.0% to about 9.0%. Accordingly,when the immersion solution does not comprise serum, concentrations ofthe serum-protein like organic molecules from about 0.03%, preferablyabout 0.08%, and greater can be used. The immersion solution canadditionally contain other biologically active molecules, as definedherein, including but not limited to attachment molecules, growthfactors, collagen, etc.

The total immersion time can vary from about 1 hour up to about twoweeks. Depending on the desired surface composition and thickness of thereaction layer(s), and concentration of serum-protein like organicmolecules, shorter or longer immersion times may be appropriate. Theimmersion times and conditions can be readily ascertained by persons ofordinary skill in the art. The differential immersion process involveseither the continuous flow of solution past the treatment surface or theperiodic replenishment of the solution. The solution is an aqueousmedium (with or without electrolytes) supplemented with serum-proteinlike organic molecules, as defined herein.

The method can be performed upon melt- or sol-gel-derived dense ormacroporous glass or ceramic with a similar result. The resultantsurface has a microporous structure, e.g., having pore sizes from about0.1 μm to about 10 μm of uniform pore size.

The immersion experiments reported herein demonstrate major differencesbetween surface modification of bioactive glass by differentialimmersion in serum-containing solutions, in comparison to those whichoccur upon integral immersion in either serum-free or serum-containingsolutions. The differential immersion in serum-containing solutionsallows one to overlay a surface layer with unique properties, i.e. tocreate a microporous Ca—P surface layer having organic moleculesintermingled throughout.

The microporous surface allows the elements of tissues to penetrate intothe micropores upon implantation. Further, the porous structurefacilitates a controlled release of molecules intermingled throughoutand, optionally, adsorbed thereon in a subsequent treatment.Additionally, the pores provide a greater surface area for interactionwith body fluids upon implantation.

The differential immersion enhances formation of crystalline Ca—P phaseswith similar characteristics as the mineral phase of bone, particularlyin comparison to the integral immersion in serum protein-containingsolutions. Indeed, FTIR analysis showed formation of crystalline Ca—Pphases after differential immersion (1 week) in serum-containingsolution (FIG. 7c). In contrast, Ca—P phases present in the surfacelayer after integral immersion for the same time period were amorphous(FIG. 10d).

In comparison, integral immersion in serum protein-containing solutionscreated a smooth, dense, fragile and amorphous surface consisting of asilica layer and a silica matrix with accumulated calcium and phosphate.

In contrast to differential immersion in serum-containing solution,differential immersion in serum-free solution produced a dense surfacelayer composed of closely packed Ca—P precipitates which cannot beintermingled with proteins.

Example 1 below describes the excavation of particles using differentialimmersion under conditions similar to the present invention. An exampleof conditioning according to the present invention, with comparison toother conditioning methods, is provided in Example 2 below.

EXAMPLE 1

Transformation of Sol-gel-derived Glass 45S5 Granules into Hollow Ca—PShells Using Differential Immersion

Specimens prepared according to Example 1 of application Ser. No.08/647,171 were immersed in three solutions: TE, TE supplemented with10% serum (vol %), and 100% serum. Newborn calf serum was used. Thesolutions were chosen to address effect of important constituents of thein vivo milieu. Immersions were performed in vials. TE is anon-proteinaceous control that contains the electrolyte constituents ofhuman blood plasma in similar concentrations (cf. Table I). TE wasprepared by dissolving reagent grade NaCl, KCl, NaHCO₃, MgCl₂.6H₂O,MgSO₄.7H₂O, KHPO₄anh. and CaCl₂.2H₂O in a 0.05 M Tris[hydroxymethyl]aminomethane hydrochloride buffered solution. The resulting pH was 7.4at 37° C.

TABLE I Ionic content of human blood plasma and TE Human blood TE Ionplasma (mM) (mM) Ca²⁺ 2.5 2.5 HPO₄ ²⁻ 1.0 1.0 Na⁺ 142.0 152.0 Cl⁻ 103.0136.0 K⁺ 5.0 5.0 Mg²⁺ 1.5 1.5 HCO₃ ⁻ 27.0 27.0 SO₄ ²⁻ 0.5 0.5

A differential immersion was effected by exchange with fresh solution atvarious time points throughout the duration of immersion. In the presentexample, the samples were exposed to fresh solution after 3, 6, 9, 24,48, 72, 96, 124 and 168 hours of immersion. These intervals were chosenin an attempt to maintain a maximum concentration of Si in solution lessthan 2/3 of the saturation concentration. The immersion protocol wasintended to reflect the continuous replenishment of body fluid at theimplant site. The samples were immersed for up to 7 days.

Three samples were tested per set of conditions. The weight to solutionvolume ratio was 0.5 mg/ml. The samples were placed in an incubator at37° C. in a 5% CO₂ atmosphere and continuously shaken (200revolutions/minute). The vials were loosely capped to minimizeevaporation without preventing gas exchange.

The testing conditions (i.e., sample, immersion mode, and solution) andthe parameters studied are listed in Table II. Up on completion of theselected immersion protocols, the solutions were collected and theretrieved particles were rinsed with ethanol and dried in ambient air.

TABLE II Testing conditions Composition Particle size Solution S100500-710 μm TE S100V S70 S70 210-500 μm TE S70 500-710 μm TE TE + 10%serum serum

The Si and Ca concentrations were measured by flame atomic absorptionspectrophotometry (FAAS, Perkin-Elmer 5100PC). The P concentration wasdetermined using a colorimetric method (Heinoken et al., “A new andconvenient colorimetric determination of inorganic orthophosphate andits application to the assay of inorganic pyrophosphate,” Anal.Biochemistry, 113:313-7, 1981,) (Molybdenum yellow) (Pharmacia LKBUltrospec Plus Spectrophotometer).

The cumulative variation of the Si, Ca, and P concentration in TE as afunction of time of immersion is shown in FIGS. 1a-c. The error barsrepresent the standard deviation of the means. As is evident from FIG.2a, 80% of the initial silicon content is released from the S70 glassafter 48 hours of immersion and no silicon remained after a 7-dayperiod. FIGS. 1b and 1 c show that the Si dissolution from S70 isaccompanied by the formation of a Ca—P phase, as indicated by acontinuous calcium and phosphate uptake from solution. For S100 glass,60% of the initial silicon was released after 72 hours and, again, nosilicon remained after a 7-day period. However, Si dissolution from S100was not accompanied by a formation of a Ca—P phase, as indicated by thelack of uptake of calcium and phosphate from solution. The presence ofVancomycin in S100 did not affect the dissolution behavior of thecomposite, as indicated by the similar release profiles.

To determine the immersion-induced compositional and structural changes,the reacted particles were analyzed with the same techniques as used forthe characterization. In addition, scanning electron microscopy incombination with energy dispersive x-ray analysis (SEM-EDXA, JEOL T300A)was employed.

FTIR analysis confirmed the formation of a calcium-phosphate materialwith the characteristics of a crystalline, carbonated hydroxyapatitesimilar to bone-mineral Ca—P.

SEM micrographs of S70 reacted particles show that the particles aretransformed into Ca and P containing shells (see FIG. 2, X 750). Thesample was prepared for SEM examination by fracturing it. As a result,the interior of the particle was exposed. The SEM examination revealedthat the particle had been transformed into a shell by the immersiontreatment.

EDXA analysis only detected calcium and phosphorus, but no silicon. TheCa/P ratio of the shell as measured by EDXA is 1.5. The shell wasapproximately 10 μm thick.

FIG. 3 shows the silicon release profile S70 particles of two sizesranges. These results indicate that the dissolution rate increases withdecreasing particle size or increasing external surface area. Moreover,the S70 particles of the smaller size range exhibit the sametransformation into shell-like shape as found for the larger particles.

FIG. 4 shows the dissolution behavior of S70 in TE, TE supplemented with10% serum, and serum. The presence of serum in the immersion solutiondecreased the rate of dissolution. Statistical analysis revealed thatthe release profiles differed significantly among solutions (p<0.05,analysis of variance) except for TE supplemented with 10% serum and 100%serum.

EXAMPLE 2

Conditioning of Bioactive Glass Surfaces

A) Modification of melt-derived glass 45S5 granules by differentialimmersion in serum-containing solution

Surface modification of melt-derived bioactive glass 45S5 granuleshaving a size of about 300 to about 355 μm as obtained by sieving wasconducted by differential immersion in Tris buffered (pH 7.4 at 37° C.)solution supplemented by electrolytes typical for plasma (Table I) and10% newborn bovine serum (TES) for up to 1 week. The samples wereimmersed at 1 mg/ml weight-to-solution (W/V) ratio. The solution wasexchanged at 3, 12, 24, 48, 96 and 168 hours. The post-immersion sampleswere dried and proceeded for the surface analysis.

Morphology, composition and structure of the surfaces, modified bydifferential immersion in serum-containing solution, are shown on theSEM micrograph and corresponding EDXA and FTIR spectra (FIGS. 5a-c).

SEM micrograph (FIG. 5a) of the surface indicates that differentialimmersion in serum-containing solution led to formation of a microporoussurface on top of dense glass. The surface layer comprises very fine(0.2 to 1 μm) precipitates. The pore size varies from 0.5 to 3 μm. Thesurface was mainly composed of Ca—P phases with addition of Si (FIG.5b). The presence of the Ca—P phases is confirmed by FTIR analysis (FIG.5c). Split of the P—O bands, recorded on the FTIR spectrum, suggeststhat the Ca—P phases were crystalline. The appearance of the C═O bandindicates the presence of adsorbed proteins in the surface layer.

FIGS. 6a-c depict an SEM of a cross-section of a granule treated bydifferential immersion as described above (FIG. 6a), full EDXA spectraof the cross-section (FIG. 6b), and extended carbon peaks from the EDXAspectra. The EDXA spectra were taken in three spots located either onthe outer side (O), in the middle of (M), or on the lower side, i.e.,inner side, (L), of the granule. As is clear from these spectra, carbonis present throughout the particle. These results indicate that theserum proteins are present throughout the Ca—P layer, which is 25-30microns thick in FIG. 6a.

B) Modification of melt-derived glass 45S5 granules by differentialimmersion in serum-free solution

Bioactive glass samples as described in A) were immersed differentiallyin serum-free Tris buffered solution complemented by electrolytestypical for plasma (TE) for up to 1 week. The samples were immersed at 1mg/ml W/V ratio. The solution was exchanged at 3, 12, 24, 48, 96 and 168hours. The post-immersion samples were dried and proceeded for thesurface analysis.

Morphology, composition and structure of the immersion-modified surfaceare shown on FIGS. 7a-c.

SEM micrograph (FIG. 7a) shows the appearance of the reaction surfacelayer (central and left parts of the picture) on top of the fracturedsample. The layer appears as dense and composed of closely packedglobular precipitates. The EDXA (FIG. 7b) indicates that theprecipitates comprise Ca—P phases. No significant Si-content wasdetected in the surface layer. Split of the P—O bands, recorded on theFTIR spectrum (FIG. 7c), suggests that the Ca—P phases were crystalline.

C) Modification of melt-derived glass 45S5 granules by integralimmersion in serum-free solution followed by immersion inserum-containing solution

Bioactive glass samples as described in A) were immersed integrally (nosolution exchange) in serum-free Tris buffered solution complementedwith electrolytes typical for plasma (TE) for 48 hours and thenre-immersed in serum-containing TES for either 1, 3 or 6 hours. Afterimmersion the samples were dried and proceeded for surface analysis.

Morphology, composition and structure of the bioactive glass surfaceafter immersion in TE for 48 hours and subsequent immersion in TES for 1hour are shown on FIGS. 8a-c. Extension of immersion in TES for up to 6hours did not produce a significant change in the surface morphology andcomposition.

SEM micrograph (FIG. 8a) indicates that the reaction surface appears asa smooth, dense layer with patchy globular precipitates (cracks areartifacts due to drying). EDXA reveals that the surface layer has amajor Si-content with addition of Ca and P (FIG. 8b). The appearance ofan undivided P—O band on the FTIR spectrum suggests the Ca—P phases wereamorphous.

The smooth reaction layer is composed of silica-matrix with Ca—Paccumulations in it. Subsequent to formation of this layer Ca—Pprecipitation occurs on top of it.

A Rutherford backscattering spectrum (RBS) of a bioactive glass diskimmersed as described above (i.e., integrally for 48 hours in TEfollowed by immersion in tissue culture medium containing 10% NU serumfor one hour) is depicted in FIG. 9. The sample was washed afterimmersion by using ethanol and acetone, and then air dried. The depthprofile for carbon and nitrogen showed the adsorption of about 1000monolayers of proteinaceous material onto the Ca—P layer. The surfaceresolution of RBS is very high. Accordingly, probing depth is typicallylimited to about 0.1 to 1 micrometer. The results indicate, therefore,that the proteinaceous material is concentrated at the surface aftersuch treatment and not throughout the Ca—P layer.

D) Modification of melt-derived glass 45S5 granules by integralimmersion in serum-containing solution

Bioactive glass samples as described in A) were immersed integrally (nosolution exchange) in serum-containing TES for 1 week. After immersionthe samples were dried and proceeded for the surface analysis.

Morphology, composition and structure of the immersion-modified surfaceare shown on FIGS. 10a-d.

SEM micrograph (FIG. 10a) shows that the reaction surface appears as asmooth and dense layer (cracks are artifacts due to drying). Noprecipitation was observed on top of this layer. EDXA spectrum on FIG.10b indicates that the layer has a major Si-content (50%, atomic) withaddition of Ca and P. The underlying layer, exposed in cracks, is mainlycomposed of silica (FIG. 10c). The appearance of an undivided P—O bandon the FTIR spectrum (FIG. 10d) indicates that the Ca—P phases wereamorphous.

The smooth reaction layers, formed on top of bioactive glass 45S5, arecomposed of silica-matrix with amorphous Ca—P accumulations in it.

The foregoing examples are meant to illustrate the invention and not tolimit it in any way. Those skilled in the art will recognize thatmodifications can be made which are within the spirit and scope of theinvention as set forth in the appended claims.

All references cited herein are hereby incorporated by reference intheir entirety.

What is claimed is:
 1. A material comprising silica-based glass orceramic comprising at least 39% silicon dioxide wherein the surface ofsaid glass or ceramic has been conditioned by: a) immersing said glassor ceramic in an aqueous solution containing serum-protein like organicmolecules for a period of time sufficient to form a microporouscalcium-phosphate (Ca—P) surface layer having serum-protein like organicmolecules intermingled throughout, and b) exchanging the solution duringsaid period of time at intervals sufficient to allow continuousformation of the microporous Ca—P layer; wherein said conditionedmaterial has a microporous Ca—P surface layer having serum-protein likeorganic molecules intermingled throughout.
 2. The material according toclaim 1 wherein said microporous surface layer has pores having apore-size from about 0.1 μm to about 10 μm.
 3. The material according toclaim 1 wherein said glass or ceramic is bioactive.
 4. The materialaccording to claim 1 wherein said glass or ceramic is macroporous. 5.The material according to claim 1 wherein said glass or ceramic isdense.
 6. The material according to claim 1 wherein said microporoussurface layer further comprises biologically active moleculesintermingled throughout.
 7. The material according to claim 1 or 6,further comprising biologically active molecules adsorbed thereon. 8.The material according to claim 1 wherein said microporous Ca—P surfacelayer further comprises silicon.
 9. A material comprising silica-basedglass or ceramic comprising at least 39% silicon dioxide, and having amicroporous Ca—P surface layer having serum-protein like organicmolecules intermingled throughout, wherein said microporous surfacelayer has pores having a size from about 0.1 μm to about 10 μm.
 10. Thematerial according to claim 9 wherein said glass or ceramic isbioactive.
 11. The material according to claim 9 wherein said materialis macroporous.
 12. The material according to claim 9 wherein saidmaterial is dense.
 13. The material according to claim 9 wherein saidmicroporous surface layer further comprises biologically activemolecules intermingled throughout.
 14. The material according to claim 9or 13 wherein said microporous Ca—P surface layer further comprisesbiologically active molecules adsorbed thereon.
 15. The materialaccording to claim 9 wherein said microporous Ca—P surface layer furthercomprises silicon.